Mass spectrometry protease selection

A specific application of this last method is the identification of epitopes recognized by a given antibody [155-157]- As illustrated in Figure 8.25, two possible approaches are used. In the first one, the protein presenting the epitope is digested by a protease and the peptides obtained that present the epitope are selected by the antibody. The mass spectrometry... 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

Any proteomic study starts with the collection of proteins from biological samples such as cell culture media, cultured cells, serum, or any biological fluid, and a variety of animal tissues. The first step is to obtain a protein sample under conditions of least protein degradation. This involves use of various protease inhibitors that stop the protein degradation. The use of protease inhibitors depends on the type of sample and the analytical technique used in the subsequent analysis. The selection of protease inhibitors used is critical since many protease inhibitors and detergents used in the preparation of tissue homogenates can interfere with mass spectrometry (MS)... 

---